Salmonella enterica serovar Typhimurium (Salmonella Typhimurium) is one of the most common causes of enterocolitis in humans. Pathogenesis of this facultative intracellular pathogen is dependent on the ability to invade non-phagocytic cells, such as those found in the intestinal epithelium. Invasion is dependent on a type III secretion system (T3SS1), which is used to translocate a set of bacterial effector proteins into the host cell. Following internalization, intracellular Salmonella survive and replicate within a modified phagosome, the Salmonella-containing vacuole (SCV). A second type III system (T3SS2) is induced intracellularly and is associated with intracellular survival/replication and biogenesis of the SCV. To understand Salmonella pathogenesis we must dissect the roles of the individual T3SS1 and T3SS2 effector proteins as well as the mechanisms that control their expression and activity inside host cells. Since the expression and function of these virulence factors is exquisitely dependent on the intracellular environment, we are focusing on developing appropriate in vitro systems to study their activities at the molecular level. We are currently using a both primary human macrophage model system and polarized epithelial cells. We have also collaborated with Garry Adams at Texas A&M to look at the involvement of Salmonella-host cell interactions in the bovine intestinal epithelium in vivo. A separate part of this project is to develop novel tools for studying effector function. One approach we have taken is to raise monoclonal antibodies by injecting mice with pooled effector proteins. Approximately half of the monoclonals that we have characterized are specific for the actin-binding effector SipA. A peptide scanning method has been used to identify the epitopes recognized by these antibodies. The epitopes are located within all three characterized domains of SipA, including the actin binding domain. We have also screened the anti-SipA antibodies for by immunofluorescence microscopy, ELISA and immunoprecipitation. And these screens indicate that within this group of 19 antibodies there are ones that will be useful for specific techniques. For example, three of the antibodies specifically detect translocated SipA in the cytosol of Salmonella-infected HeLa cells. Most of the other antibodies recognize known effector proteins but some recognize proteins that have not been identified as T3SS1 effectors. We are also developing new molecular biology tools, for example using tunable synthetic promoters or inducible promotors to study the functions of individual virulence genes.